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Here we demonstrate the HaloTag® Protein reporter fused to truncated human CD59 at the amino terminus can be efficiently expressed on the extracellular surface of mammalian cells. Using a non-permeable HaloTag® Alexa488 Ligand and a permeable HaloTag® TMR Ligand, we were able to separate the surface pool and the intracellular pool of the fusion protein. We were also able to monitor bidirectional trafficking of the HaloTag® GPI fusion chimera in real time and demonstrate the effect of different conditions on trafficking. Data of immunocytochemical analysis using anti-HaloTag® antibody confirmed our live cell imaging results. Fluorescently labeled protein pools were further analyzed using SDS-PAGE, fluoroimaging, and Western blot. As was expected, only the fusion proteins containing the N-terminal signal peptide and the proper GPI-anchoring sequence can be labeled with both cell-permeant and -impermeant ligands. Taken together our results indicate that the HaloTag® Technology can be used to study trafficking of GPI-anchored proteins and can be applied to both life science research and potentially drug development research.

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